Immunohistochemistry (“IHC”) is a method for testing cells or tissue samples for the presence of specific molecules. These methods are useful in both research and diagnostic applications for analyzing cell or tissue specimens. For example, in a diagnostic setting the molecular profile of a tissue can provide evidence of a particular disease state, such as cancer. These IHC methods first comprise the step of preparing an antibody to the particular molecule of interest. An antibody is a protein that is made to specifically recognize and combine with a molecule of interest. The molecule of interest is referred to as an antigen, and an antibody can specifically recognize and bind to its antigen. The specificity of this reaction allows an investigator to infer the presence of the antigen (target molecule, or target) whenever the antigen-antibody reaction takes place. For example, in the diagnosis of cancer, a specific antibody to a cancer-associated antigen is placed in contact with cells or tissue sample suspected of being cancer, If the antigen-antibody reaction occurs, this indicates that the suspected tissue was in fact cancer,
The steps of IHC typically include fixing a tissue by placing a tissue into a fixative such as formalin. The fixative has two primary effects, First, it rapidly stops all metabolic activity in the cells so there is no degradation of molecular structures or changes in morphology, and secondly it makes the tissue rigid so that it can be embedded in hot paraffin and still retain its overall structure. Fixation with formalin is accomplished by inducing chemical cross-linking within and between molecules, particularly protein molecules. Other suitable fixation methods may also be used,
Next, the fixed tissue is embedded in hot paraffin which is allowed to cool to form a solid paraffin block containing the embedded tissue. The paraffin block is then cut into thin slices of the tissue. A thin tissue section is applied onto a microscope slide such that the tissue can be subsequently examined under the microscope once the staining reaction has been completed.
The microscope slide may be treated with the attached tissue with a processing fluid that contains a paraffin solvent, such that the paraffin that is used to embed and mount the tissue onto the microscope slide is removed. Then the microscope slide is typically treated with a series of alcohols to remove the paraffin solvent, finally the microscope slide is treated with a series of aqueous solutions to rehydrate the tissue back into its native state. These steps, called deparaffinization, effectively remove the paraffin while leaving the tissue rehydrated and still adherent to the microscope slide.
The next step typically involves applying an antigen retrieval (“AR”) solution to expose antigens within the tissue. In the process of fixing the tissue, the molecular structure of the protein is frequently altered such that an antibody reagent will no longer react with its target r molecule. In order to overcome this limitation, AR methods were developed that have the ability to reverse the cross-links and restore the molecules to a more native configuration that can be recognized by the antibody reagent. This step of the process is called heat-induced AR, or simply AR.
The tissue is then typically treated on a microscope slide with a chemical to block endogenous enzyme activity. A specific (primary) antibody may be applied onto a tissue sample. The primary antibody is allowed enough time to bind to its antigen (if present). The bound primary antigen may be visualized by adding an enzyme linked to a secondary reagent. A substrate/chromogen is added which reacts with the enzyme to form a colored (dye) end-product. The colored end-product is visualized by viewing the tissue sample under the microscope. If the colored end-product is observed, then the tissue contained the suspected antigen. If no colored end-product is observed, then the tissue did not contain the suspected antigen.
Another method commonly used for studying cells or tissues is called In Situ Hybridization (“ISH”). In this method the target that is being analyzed is a nucleic acid (either DNA or RNA). The probe that is used to detect the nucleic acid is a complementary strand of nucleic acid. The rationale behind this approach is that complementary strands of nucleic acids will bind to each other. Therefore, it is possible to synthetically construct a probe with a complementary nucleic acid sequence to the target nucleic acid. The target nucleic acid may be a gene or gene fragment, it may be nucleic acid of a bacterial or viral pathogen, or it may be a gene product, such as an mRNA. The binding of a probe to a target nucleic acid is indicative of the presence of the target nucleic acid in the cells or tissues under investigation. For example, the amplification of a gene called Her2/neu is a primary driver for the development of certain breast cancers. By applying a probe to Her2/neu gene in a breast cancer specimen, it can be determined whether that breast cancer had an abnormal amplification (>2) of the Her2/neu gene or whether that breast cancer had a normal copy number (=2) of the Her2/neu gene. This information can then be used to direct the most optimal therapy.
The steps of ISH typically include fixing a tissue by placing a tissue into a fixative such as formalin. The fixed tissue is then embedded in hot paraffin which is allowed to cool to form a solid paraffin block containing the embedded tissue. Thin slices of the tissue are cut from the paraffin block. A thin tissue section is then applied onto a microscope slide such that the tissue can be subsequently examined under the microscope once the staining reaction has been completed.
The microscope slide is treated with the attached tissue to a processing fluid that contains a paraffin solvent, such that the paraffin that is used to embed and mount the tissue onto the microscope slide is removed. The microscope slide is treated with a series of alcohols to remove the paraffin solvent, and the microscope slide is treated with a series of aqueous solutions to rehydrate the tissue back into its native state. These steps call deparaffinization, effectively remove the paraffin while leaving the tissue rehydrated and still adherent to the microscope slide.
The next step involves applying a target retrieval (“TR”) solution to expose nucleic acids within the tissue. In the process of fixing the tissue, the molecular structure of the nucleic acids is frequently altered such that a probe reagent will no longer react with its target molecule. In order to overcome this limitation TR methods were developed that have the ability to reverse the cross-links and restore the molecules to a more native configuration that can be recognized by the probe reagent. This step of the process is called heat-induced TR. In another related procedure the tissue may be treated with an enzyme that accomplishes essentially the same thing as the heat-induced TR. If this process is utilized it is referred to as enzyme pretreatment or enzyme TR.
A tissue on a microscope slide may then be treated with a chemical to block endogenous enzyme activity. A specific probe may be applied onto a tissue sample that has a nucleic acid sequence complementary to the target being detected. The probe is allowed enough time to bind to its target (if present). The bound probe is visualized by adding an enzyme linked to a secondary reagent. A substrate/chromogen is added which reacts with the enzyme to form a colored (dye) end-product. The colored end-product is visualized by viewing the tissue sample under the microscope.
The use of the capillary gap method for processing slides is riot new to the field of immunohistochemistry. In the late 1980's David Brigati (U.S. Pat. No. 4,731,335) described the adaptation of an automated instrument that was designed to remove paraffin from tissue samples and to rehydrate the tissue samples. This device held a number of microscope slides vertically in a slide holder which were then submerged into a series of reagent baths to first dissolve the paraffin and then to rehydrate the slides in preparation for immunohistochemistry. In Brigati's adaptation, a custom microscope slide with raised spacers was needed where the slides were in mounted in the vertical slide holder in pairs facing each other, so that a capillary gap was formed between the two adjacent facing slides. Instead of submerging the slides into a bath, the bath system was adapted to form shallow troughs that contained only a few hundred microliters of reagents. When the ends of the slide pairs contacted the reagent troughs, the liquid was drawn up between the slide pairs by capillary action. Likewise, in order to remove reagents the ends of the slide pairs were brought into contact with an absorbent material which caused a reverse capillary flow and withdrew the reagents from the capillary space. Major drawbacks of the Brigati method were 1) there was a requirement for a specialized slide that contained raised areas to form the capillary space (the available commercial microscope slides were not suitable for the Brigati method), and 2) the resultant staining was not uniform. Typically the bottom portion of the specimen stained darker than did the top part of the specimen. The uneven staining was the result of lack of mixing of the processing fluids within the capillary space.
For the foregoing reasons there is a need for a new system for processing biological specimens that can utilize standard, economical microscope slides, while producing consistent results.